Project Summary/Abstract Oxysterols, the oxygenated derivatives of cholesterol, have been identified for several decades, however, their roles in the physiology and pathological processes, particularly in nervous system, are largely unknown. Our previous study showed that the major cholesterol metabolite, cholestane-3?, 5?, 6?-triol (Triol), functions as an important endogenous neuroprotectant. Here, we hypothesize that blockade of acid-sensing ion channel (ASIC) 1a serves as a novel mechanism underlying its neuroprotective effect, based on the following arguments and preliminary data: (1) The concentration of Triol increases after ischemic preconditioning (IPC), which contributes to IPC mediated neuroprotection; (2) The activation of calcium permeable ASIC1a exacerbates ischemic injury, and that pharmacological blockade or knockout of ASIC1a produces a significant protection; (3) In brain ischemia, blockade of ASIC1a has a therapeutic time window of ~5h, which is close to that of Triol (> 4h); (4) Our preliminary data clearly demonstrated that Triol significantly inhibits ASIC1a currents as well as its surface expression, at a concentration that occurs during ischemic preconditioning; (5) The preliminary data suggest that the inhibition of ASIC is specific to Triol, as its precursor cholesterol has no effect on ASIC currents. To rigorously test the hypothesis that Triol functions as an endogenous neuroprotectant through blockade of ASIC1a, the following specific aims will be examined: Aim 1. Determine the effect of Triol on ASIC current and calcium entry. Aim 2. Determine the inhibitory effect of Triol on ASIC1a surface expression. Aim 3. Determine whether the neuroprotection caused by Triol is mediated through inhibition of ASIC1a. Aim 4. Study the structure-activity relationship, and identify the amino acids that mediate the inhibitory effect of Triol on ASIC1a.